1α,25‐Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells

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Abstract
1α,25-Dihydroxyvitamin D3 increases intracellular calcium in rat osteoblast-like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25-dihydroxyvitamin D3 (20 nM). This effect was blocked by the inactive epimer 1β,25-dihydroxyvitamin D3. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25-dihydroxyvitamin D3, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25-dihydroxyvitamin D3, 20 nM, had no effect on nuclear calcium but blocked the 1α,25-dihydroxyvitamin D3 induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25-dihydroxyvitamin D3. 1α,25-Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity.

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